This project involves the development and application of a system for the real-time analysis of clathrin-mediated endocytosis in living cells utilizing total internal reflection fluorescence microscopy (TIR-FM) TIR-FM represents a microscopy technique capable of selectively imaging events occurring adjacent to the plasma membrane with very high sensitivity. Clathrin mediated endocytosis is responsible for the internalization of activated receptors and ligands, nutrients, and cell adhesion molecules. Several genetic diseases are caused by defects in endocytosis (e.g. familial hypercholesterolemia) and numerous pathogens (e.g. influenza virus) gain entry into cells by exploiting the endocytosis machinery. Although many proteins involved in clathrin mediated endocytosis have been identified, the current techniques for studying endocytosis lack a real-time kinetic analysis of the formation and internalization of individual coated pits and of the interactions among the proteins involved. TIR-FM has the potential to permit analysis of clathrin mediated endocytosis in living cells and allows simultaneous imaging of the proteins involved the formation and fission of clathrin-coated pits under various physiological conditions (e.g. migrating cells, polarized cells and specialized cells).